The impact of bilirubin ditaurate on platelet quality during storage

Abstract

HPLC MethodBriefly, HPLC-PDA analysis was performed using a Shimadzu Prominence HPLC system consisting of an online degassing unit (DGU20A5R), solvent delivery unit (LC-20AT), autosampler (SIL-20 AC), column oven (CTO-20) and photo diode-array (SPD-M20A), connected in series. Separation was achieved via reverse phase C18 column (GraceSmart™ RP-C18, 150mm x 4.6mm, 3µm; Grace Davidson, Australia), which was preceded by a guard column (GraceSmart™ RP-C18, 3µm; Grace Davidson, Australia) and an UltraLine HPLC in-line filter (Restek, 0.5µm; Shimadzu, Sydney, Australia), respectively. The column oven and autosampler were set to 45˚C and 4˚C, respectively, with an initial flow rate of 1.6mL•min −1 . Starting mobile phase consisted of 20% organic B (100% HPLC grade MeOH) and 80% aqueous A (10mM NH4OAc in 25% HPLC grade MeOH and 75% Milli-Q purified H2O). A linear gradient was applied, reaching 90% B at 4.5 minutes, remaining until 7 minutes. From 7 to 11.5 minutes, the mobile phase was set at 20% B. To assist equilibration, flow rate remained at 1.6mL•min −1 until 6 minutes where it increased to 2.3mL•min −1 at 6.35 minutes, remaining until 8.5 minutes. From 8.5 to 9.5 minutes, the flow rate decreased to 1.6mL•min −1 and remained until 11.5 minutes. The total run time including adequate column re-equilibration was 11.5 minutes. For UCB and BRT extraction, 160μL of extractant (1:4 DMSO:MeOH) was added to 40μL of PPM sample, mixed via vortex for 10 seconds before centrifugation (21 500RCF; 10 minutes) to pellet protein. The supernatant was diluted (2:1) with Milli-Q H2O upon addition to a HPLC vial, prior to being placed in the autosampler where 40µL was injected for analysis. Commercially prepared BRT and UCB (Frontier Scientific Inc. Logan, UT, USA) served as external standards with retention times of 4.1 and 5.1 minutes respectively. Extraction efficiencies of these compounds from PPM was 95% and 91% for BRT and UCB, with standard curves indicating great linearity over concentration ranges of 1.5625µM -100µM (r 2 =0.9993) and 0.15625µM -10µM (r 2 =0.9995)